TKTL1 Immunohistochemistry in Metastatic Melanoma

Immunohistochemistry was performed on tissue microarrays constructed from formalin-fixed paraffin-embedded (FFPE) metastatic melanomas and melanoma cell lines after citrate buffer (pH 6.0) antigen retrieval for 30 min using the Dako Envision⁺ kit (Dako). Incubation with 1× protein blocking buffer for one hour at room temperature was followed by overnight incubation at 4 °C with mouse monoclonal anti-TKTL1 antibody (clone JFC12T10; 1:200 dilution) previously described by Langbein et al. [17 (link)]. 60 min incubation with secondary anti-mouse antibody HRP (Dako) was performed. 3-amino-9-ethylcarbazole (AEC) was used as chromagen (Vector Laboratories). Slides were counterstained with hematoxylin, images were captured and immunohistochemical reactivity was evaluated by two independent investigators. Patient tumor cores were scored TKTL positive based on cytosolic staining in minimum of 10 % tumor cells. Human testis tissue was used as the positive control for TKTL1 and a negative control, for which the primary antibody was substituted with the same concentration of mouse IgG.